Benzodiazepines are normally prescribed for therapeutic use, as tranquilizers, muscle relaxants, hypnotics, sedatives, treatments for insomnia, anticonvulsants, and the like. The variety of these drugs and their level of consumption by the public is increasing at a fast rate. They frequently become drugs of abuse, however, and can easily reach toxic levels when administered for purposes other than therapeutic. For this reason, it is frequently necessary to determine the presence, type and amount of benzodiazepines in samples of urine and other biological fluids.
A common method of analysis for benzodiazepines and benzodiazepine metabolites is thin-layer chromatography of urine samples. Other methods include high-performance liquid chromatography and gas chromatography. These analytical methods are complicated however by a number of factors. One of these is the fact that the primary benzodiazepine metabolites (i.e., glucuronides) are extremely polar compounds and are not easily extracted from biological fluids. Extraction can be facilitated by first reacting the samples with .beta.-glucuronidase, but this reagent is not recovered during the analysis, and the reaction typically requires from 4 to 16 hours to complete. Also, the concentrations of analytes in biological fluids after therapeutic doses can be very low, in some cases less than 10 ng/mL, and as a result, extremely sensitive detectors are necessary in order to reduce the possibility of false negative results. A further difficulty is that most benzodiazepines have very similar chemical structures, and are difficult to separate using standard chromatographic techniques. Still further, it is difficult to identify the compounds based solely on data such as UV spectra and gas chromatography-mass spectroscopy data which is generated by typical chromatograph detectors. To summarize the problems encountered in benzodiazepine detection and identification, these are:
(1) labor intensive sample preparation; PA1 (2) difficulty in achieving adequate separation of all compounds in the class; PA1 (3) difficulty in achieving adequate sensitivity and specificity; PA1 (4) high-cost detection systems; and PA1 (5) the requirement for high levels of technical expertise.